Thiol isomerases are critical for disulfide bond formation during protein synthesis within the endoplasmic reticulum, but protein thiol isomerases located outside of the endoplasmic reticulum may be critical for regulation of redox-sensitive allosteric disulfide bonds in proteins. Thiol isomerases in platelets and endothelial cells may play an important role in the regulation of protein function during thrombus formation. Our hypothesis is that each thiol isomerase that putatively influences thrombus formation in vivo has a specific function on a specific substrate(s). Protein disulfide isomerase and members of the PDI family will be identified in resting platelets and unstimulated endothelial cells, and localized to either the cell surface or internal organelles, or both, by flow cytometry, immunofluorescence microscopy and electron microscopy. The redistribution of thiol isomerases during platelet activation and endothelial cell stimulation, with special focus on the release of the enzymes and their binding to the cell surface, will be explored. To identify potential thiol isomerase substrates, proteins expressing free thiols on the surface of platelets and endothelial cells will be labeled, the membranes purified, and these proteins separated by 2D gel electrophoresis using 16-BAC/SDS. These labeled membrane proteins will be identified by mass spectrometry and will represent possible thiol isomerase substrates. To determine the role of thiol isomerases during thrombus formation, the kinetics of expression of thiol isomerases, including PDI, ERp5, ERp54, ERp72, ERp46, ERdj5, TMX and TMX3 will be studied in a live mouse by intravital multichannel widefield and confocal microscopy. The effect on inhibition of each thiol isomerase using antibodies specific for thiol isomerases on platelet activation monitored by calcium mobilization, platelet alpha granule release detected by P-selectin expression, fibrin formation, thrombin activity, platelet accumulation, platelet on/off rates, platelet thrombus embolization, microparticle accumulation, vWF accumulation, TF expression, and collagen exposure will be evaluated in the laser-induced vessel wall injury model and the ferric chloride model in a living mouse. These studies are designed to determine the role of thiol isomerases released from platelets and endothelial cells, and their contribution to the modulation of function of proteins involved in thrombosis and hemostasis, including but not limited to GPIb, GPIIbIIIa and tissue factor.